The present study is based on three central aspects (i) Evaluation of total toxic elements (TEs) i.e. arsenic (As), copper (Cu), cadmium (Cd), lead (Pb) and nickel (Ni) in smokeless tobacco (SLT) products (mainpuri, mawa, gutkha, dry and moist snuff), available and consumed in Pakistan, (ii) Method development for the assessment of As, Cd, Pb, Ni and Cu in artificial saliva extract (ASE) of different types of SLT products, (iii) To assess the contents of studied TEs in biological samples of oral, pharyngeal cancer patient and adolescent boys who have consumed SLT (mawa, mainpuri, gutkha, moist and dry snuff) products. ❖ To determine the trace levels of As, Pb, Cd, and Ni concentration in ASE of SLT products, simple and rapid pre-concentration methods were developed such as cloud point extraction, dual-cloud point extraction, temperature-controlled ionic liquid-based ultrasound-assisted dispersive liquid–liquid microextraction, non-dispersive ionic liquid based microextraction, followed by flame (FAAS) and electrothermal atomic absorption spectrometry (ETAAS). Total inorganic arsenic (iAs) and As3+ in ASE of SLT were determined by solid phase and cloud point extraction methods, respectively. ❖ The artificial saliva can extract studied TEs (As, Cd, Cu, Ni and Pb), which is immediately available, absorbed and affecting the lining of mouth. The artificial saliva extracted As, Cd, Cu, Ni and Pb corresponds to 15 - 24, 39 - 75, 30 - 50, 21 - 58, and 18 - 32%, respectively, of total contents of each TEs in different SLT (mawa, mainpuri, gutkha, dry and moist snuff) products. ❖ To determine the studied TEs concentrations in biological samples (scalp hair, blood), advance extraction methodologies were developed. The innovative vortex-assisted liquid– liquid microextraction and ionic liquid based microextraction in a single syringe system have been developed, prior to analysis by FAAS. The main factors which affect the recoveries of trace elements were studied. The detection limit and enhancement factor were also calculated at optimized values of significant factors. ❖ The reliability of methodologies was assured by analyzing virginia tobacco leaves (ICHTJcta-VTL-2), BCR 397 of human hair, Clincheck control-lyophilized® human serum SeroM10181 and whole blood Recipe (certified reference materials). ❖ The contents of As, Cd, Pb and Ni in scalp hair and blood samples were measured in three different adolescent (boys) subgroups consuming different SLT products alone or in conjunction with each other. The results revealed that adolescents who have used SLT have 2-3 folds higher contents of As, Cd, Pb and Ni in their biological samples than referent boys (p<0.001). ❖ The relationship between As, Cd, Ni and Pb exposure in oral cancer patients via consumption of SLT products with related to referents were investigated. The Cd, As, Pb and Ni in SLT products and biological samples (blood and scalp hair) of cancerous and healthy subjects were evaluated. The referents and oral cancer patients have similar age group (ranges from 30 to 60 years), localities and socio-economic status. The results showed that the Ni, As, Pb and Cd concentrations were higher in the blood and scalp hair samples of oral cancer patients related to the results obtained from referents (p<0.001). ❖ The Cu level in serum samples of oral (n=65) and pharyngeal (n=53) cancer patients (male), age ranged (30-60 years) has been assessed. For comparative study, the serum samples of referents (n=150), used/not consumed chewing SLT were also investigated for Cu level. The Cu contents in SLT and serum samples were evaluated by ETAAS. The levels of Cu in samples (serum) of oropharyngeal cancer patients were 1.5-2 and 3.0-4.0 folds higher than those referents consumed different SLT product and who had none of this habit, respectively.
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